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Article(id=1241065979711639639, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241065978004557893, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202410452, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1730131200000, receivedDateStr=2024-10-29, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773822787453, onlineDateStr=2026-03-18, pubDate=1740412800000, pubDateStr=2025-02-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773822787453, onlineIssueDateStr=2026-03-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773822787453, creator=13701087609, updateTime=1773822787453, updator=13701087609, issue=Issue{id=1241065978004557893, tenantId=1146029695717560320, journalId=1227665162245664772, year='2025', volume='52', issue='4', pageStart='577', pageEnd='768', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773822787047, creator=13701087609, updateTime=1773823194927, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241067688831808347, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241065978004557893, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241067688831808348, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241065978004557893, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=622, endPage=628, ext={EN=ArticleExt(id=1241065980063961187, articleId=1241065979711639639, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Generalized anxiety disorder and plasma exosome-specific miRNA sinduced by drinking- water arsenic exposure, columnId=1228016570660745413, journalTitle=Modern Preventive Medicine, columnName=Environmental and Occupational Health, runingTitle=null, highlight=null, articleAbstract=
Objective

To examine the impact of drinking water arsenic exposure on the development of generalized anxiety disorder (GAD) and to investigate specific changes in plasma exosomal miRNAs.

Methods

Sixteen male SPF-grade Wistar rats were randomly divided into two groups: control and arsenic. Each group consisted of 8 rats. The arsenic group was exposed to 0.10 mg/L. All groups had unrestricted access to drinking water. The rats were exposed to arsenic for a duration of 21 days. Behavioral assessments using the open field and elevated plus maze tests were conducted on days 0, 7, 14, and 21 to evaluate GAD-like behavior. At the end of the experiment, plasma exosomal miRNAs were extracted from both groups of rats. Sequencing was performed, and the expression levels of differentially expressed miRNAs were validated using real-time quantitative PCR.

Results

On the 21st day after arsenic exposure, the arsenic group exhibited lower body weight compared to the control group (t=7.950, P<0.001). In the open field test, the arsenic group showed a decrease in total distance traveled (t=2.213, P=0.044), an increase in rearing frequency (t=-4.704, P<0.001), an increase in grid crossings (t=4.340, P=0.001), and a decrease in standing frequency (t=4.496, P=0.001) compared to the control group. In the elevated plus maze test, the arsenic group exhibited a decrease in the number of entries into the open arms (t=3.614, P=0.003) and a decrease in time spent in the open arms (t=4.775, P<0.001), while showing an increase in the number of entries into the closed arms (t=-2.486, P=0.026) and an increase in time spent in the closed arms (t=-6.862, P<0.001) compared to the control group. These results indicate GAD-like behavior in the rats exposed to arsenic. Real-time quantitative PCR revealed that the expression levels of miR-99b-3p, miR-9a-5p, and miR-218a-5p were upregulated, while the expression levels of miR-425a-3p, miR-378a-3p, and miR-155-5p were downregulated in the arsenic group compared to the control group (P<0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed plasma exosomal miRNAs indicated their potential involvement in GAD development through processes such as endocytosis, MAPK pathway, Ras signaling pathway, cAMP signaling pathway, mTOR signaling pathway, and apoptosis.

Conclusion

Drinking water arsenic exposure can induce GAD-like behavior in rats. Plasma exosomal miRNAs, including miR-99b-3p, miR-9a-5p, miR-218a-5p, miR-425a-3p, miR-378a-3p, and miR-155-5p, may be involved in the regulation of GAD development induced by drinking water arsenic exposure.

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目的

了解饮水型砷暴露致广泛性焦虑障碍 (Generalizedanxietydisorder, GAD) 的作用，探索血浆外泌体miRNAs的特异性改变。

方法

将16只SPF级wistar雄性大鼠随机分到正常组和染砷组，每组8 只，染砷组饮用水中的NaAsO2浓度0.10 mg/L，两组均可自由饮水。共染毒21天，分别用旷场和高架十字迷宫实验检测染毒的第0、7、14、21天的行为学，以评估GAD样行为；实验结束后，提取两组大鼠的血浆外泌体miRNAs进行测序，并运用实时荧光定量PCR验证差异miRNAs的表达情况。

结果

染毒后的第 21 d染砷组的体重低于正常组 (t=7.950，P<0.001) 。旷场实验结果显示，与正常组相比，染砷组的总路程减少 (t=2.213，P=0.044) ，修饰次数增加(t=-4.704，P<0.001)、跨格次数(t=4.340，P=0.001)和站立次数减少 (t=4.496，P=0.001)；高架十字迷宫实验结果显示，与正常组相比，染砷组的开臂次数 (t=3.614，P=0.003) 和开臂时间(t=4.775，P<0.001)减少，闭臂次数(t=-2.486，P=0.026)与闭臂时间增加 (t=-6.862，P<0.001)，砷暴露引起了大鼠GAD 样行为。实时荧光定量 PCR 结果显示，与正常组相比，染砷组中的miR-99b-3p 、miR-9a-5p 、miR-218a-5p 表达均上调；miR-425a-3p 、miR-378a-3p 、miR-155-5p 表达均下调（P<0.05）。GO 、KEGG基因富集分析结果显示，差异血浆外泌体miRNAs可能通过内吞作用、MAPK通路、Ras信号通路、cAMP信号通路、mTOR信号通路、凋亡来参与GAD的发生发展。

结论

饮水型砷暴露可以引起大鼠的GAD样行为，外泌体miR-99b-3p、miR-9a-5p、miR-218a-5p、miR-425a-3p、miR-378a-3p、miR-155-5p可能参与调节饮水型砷暴露致GAD的过程。

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王娜，E-mail：
, copyrightStatement=本刊刊出的所有文章不代表中华预防医学会和本刊编委会的观点，除非特别声明。, copyrightOwner=中华预防医学会和四川大学华西公共卫生学院, extLink=null, articleAbsUrl=null, sourceXml=exYyI6sfZ7LSqKAWxVYBdA==, magXml=o3K8+lRcf6x8UBG4JgfZQQ==, pdfUrl=null, pdf=DCae3llFjqTRTqaW5BnGag==, pdfFileSize=1412488, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=AE4zg9d8QggxT7jI0xaGiw==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=Cx2Ly6rCWl8Sxbeu7IUiDg==, mapNumber=null, authorCompany=null, fund=null, authors=

尹紫月（1997—），女，硕士在读，研究方向：环境流行病学、精神流行病学

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尹紫月（1997—），女，硕士在读，研究方向：环境流行病学、精神流行病学

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尹紫月（1997—），女，硕士在读，研究方向：环境流行病学、精神流行病学

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Brain Research Bulletin, 2021, 174: 161-172., articleTitle=Melatonin ameliorates anxiety-like behaviors induced by sleep deprivation in mice: Role of oxidative stress, neuroinflammation, autophagy and apoptosis, refAbstract=null)], funds=[Fund(id=1241065992969835084, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, awardId=82160642, language=CN, fundingSource=国家自然科学基金(82160642), fundOrder=null, country=null), Fund(id=1241065993083081299, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, awardId=2021GXNSFBA196014; 2024JJA140974, language=CN, fundingSource=广西自然科学基金(2021GXNSFBA196014; 2024JJA140974), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241065984602198302, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, xref=1., ext=[AuthorCompanyExt(id=1241065984610586911, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, companyId=1241065984602198302, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=School of Public Health, Guilin Medical University, Guilin, Guangxi 541000, China), AuthorCompanyExt(id=1241065984618975520, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, companyId=1241065984602198302, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.桂林医学院公共卫生学院，广西 桂林 541000)]), AuthorCompany(id=1241065986070204713, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, xref=2., ext=[AuthorCompanyExt(id=1241065986078593322, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, companyId=1241065986070204713, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.东北大学生命科学与健康学院)])], figs=[ArticleFig(id=1241065990923014611, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=EN, label=Fig.1, caption=Changes in body weight of the two groups of rats at different time points, figureFileSmall=hahpHa9E/vO+2PgXHRQMqQ==, figureFileBig=AE4zg9d8QggxT7jI0xaGiw==, tableContent=null), ArticleFig(id=1241065991023677912, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=CN, label=图1, caption=两组大鼠饲料剩余量变化, figureFileSmall=hahpHa9E/vO+2PgXHRQMqQ==, figureFileBig=AE4zg9d8QggxT7jI0xaGiw==, tableContent=null), ArticleFig(id=1241065991313084901, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=EN, label=Fig.2, caption=Changes in body weight of the two groups of rats at different time points, figureFileSmall=9dkgMEi4gqKPGC73BBqyZg==, figureFileBig=eie2hqIv7h0BGtIEITRkDw==, tableContent=null), ArticleFig(id=1241065991413748206, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=CN, label=图2, caption=两组大鼠不同时点体重变化, figureFileSmall=9dkgMEi4gqKPGC73BBqyZg==, figureFileBig=eie2hqIv7h0BGtIEITRkDw==, tableContent=null), ArticleFig(id=1241065991501828593, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=EN, label=Fig.3, caption=Identification of Exosomes, figureFileSmall=I1RGy1FGfX2K06pteCAxEQ==, figureFileBig=GBF1nsUpOWrDVPN0OfuhhA==, tableContent=null), ArticleFig(id=1241065991594103288, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=CN, label=图3, caption=外泌体鉴定

注：A：透射电镜4X下外泌体形态观察，红色箭头为外泌体的经典外形-球形扁平膜囊、伴有“茶托”样；B：外泌体粒径分布统计。

, figureFileSmall=I1RGy1FGfX2K06pteCAxEQ==, figureFileBig=GBF1nsUpOWrDVPN0OfuhhA==, tableContent=null), ArticleFig(id=1241065991715738114, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=EN, label=Fig.4, caption=Expression profiles and differential expression of miRNAs in the arsenic-exposed group and the control group, figureFileSmall=vSNo94IntxqAhvKAnfaYtA==, figureFileBig=UIUcbb1l3qKakdQaUDNOUg==, tableContent=null), ArticleFig(id=1241065991828984328, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=CN, label=图4, caption=miRNAs在染砷组和正常组中的表达谱及差异表达

注：A：外泌体miRNAs种类VENN图；B：外泌体miRNAs差异表达热图；C：Real-TimePCR检测血浆外泌体差异mAiRNAs表达。

, figureFileSmall=vSNo94IntxqAhvKAnfaYtA==, figureFileBig=UIUcbb1l3qKakdQaUDNOUg==, tableContent=null), ArticleFig(id=1241065991904481807, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=EN, label=Fig.5, caption=GO and KEGG classification diagrams of predicted target genes for differentially expressed miRNAs in plasma exosomes, figureFileSmall=EPYVw1xdDNZhBij2mw63ww==, figureFileBig=jXLzwCn6lQPnO9eWv42khA==, tableContent=null), ArticleFig(id=1241065992030310930, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=CN, label=图5, caption=血浆外泌体差异miRNAs预测靶基因GO、KEGG分类图

注：A：6个差异外泌体miRNAs靶基因VENN图；B：预测靶基因GO分类图；C：预测靶基因GO分类图。

, figureFileSmall=EPYVw1xdDNZhBij2mw63ww==, figureFileBig=jXLzwCn6lQPnO9eWv42khA==, tableContent=null), ArticleFig(id=1241065992130974233, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=EN, label=Table 1, caption=

The changes of body weight at different time points for two groups of rats(,n=16,g)

, figureFileSmall=null, figureFileBig=null, tableContent=
染毒时间正常组
（n=8）		染砷组
（n=8）		t值P值
第0 d349.36±6.40351.76±7.14-0.7080.491
第7 d381.23±5.10373.90±9.441.9330.074
第14 d424.11±7.09389.76±8.548.745<0.001
第21 d469.86±17.79417.02±6.05a7.950<0.001
), ArticleFig(id=1241065992248414754, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=CN, label=表1, caption=

两组大鼠不同时点体重变化 (g,)

, figureFileSmall=null, figureFileBig=null, tableContent=
染毒时间正常组
（n=8）		染砷组
（n=8）		t值P值
第0 d349.36±6.40351.76±7.14-0.7080.491
第7 d381.23±5.10373.90±9.441.9330.074
第14 d424.11±7.09389.76±8.548.745<0.001
第21 d469.86±17.79417.02±6.05a7.950<0.001
), ArticleFig(id=1241065992344883753, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=EN, label=Table 2, caption=

The results of openfield experiment for two groups of rats()

, figureFileSmall=null, figureFileBig=null, tableContent=
组别总路程(mm)跨格次数 (次)站立次数 (次)修饰次数 (次)
正常组（n=8）28 052.791±1 743.81919.125±3.13648.625±11.8312.125±0.834
染砷组（n=8）25 923.885±2 089.08411.250±4.06224.625±9.3793.875±0.640
t值2.2134.3404.496-4.704
P值0.0440.0010.001<0.001
), ArticleFig(id=1241065992558793266, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=CN, label=表2, caption=

两组大鼠旷场实验结果 ()

, figureFileSmall=null, figureFileBig=null, tableContent=
组别总路程(mm)跨格次数 (次)站立次数 (次)修饰次数 (次)
正常组（n=8）28 052.791±1 743.81919.125±3.13648.625±11.8312.125±0.834
染砷组（n=8）25 923.885±2 089.08411.250±4.06224.625±9.3793.875±0.640
t值2.2134.3404.496-4.704
P值0.0440.0010.001<0.001
), ArticleFig(id=1241065992701399611, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=EN, label=Table 3, caption=

The results of the elevated plus maze experiment for two groups of rats()

, figureFileSmall=null, figureFileBig=null, tableContent=
组别开臂次数 (次)闭臂次数 (次)开臂时间 (s)闭臂时间 (s)
正常组（n=8）8.12±2.356.62±1.509.36±1.88173.17±13.50
染砷组（n=8）4.25±1.98.37±1.305.46±1.32237.46±22.7
t值3.614-2.4864.775-6.862
P值0.0030.026<0.001<0.001
), ArticleFig(id=1241065992797868606, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241065979711639639, language=CN, label=表3, caption=

两组大鼠高架十字迷宫实验结果 ()

, figureFileSmall=null, figureFileBig=null, tableContent=
组别开臂次数 (次)闭臂次数 (次)开臂时间 (s)闭臂时间 (s)
正常组（n=8）8.12±2.356.62±1.509.36±1.88173.17±13.50
染砷组（n=8）4.25±1.98.37±1.305.46±1.32237.46±22.7
t值3.614-2.4864.775-6.862
P值0.0030.026<0.001<0.001
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饮水型砷暴露引起广泛性焦虑障碍及血浆外泌体特异性 miRNAs研究
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尹紫月 1 , 郭林楠 1 , 雷炜星 1 , 郭雪峰 1 , 崔钧贺 2 , 余启明 1 , 张磊 1 , 王娜 1
现代预防医学 | 环境与职业卫生 2025,52(4): 622-628
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现代预防医学 | 环境与职业卫生 2025, 52(4): 622-628
饮水型砷暴露引起广泛性焦虑障碍及血浆外泌体特异性 miRNAs研究
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尹紫月1, 郭林楠1, 雷炜星1, 郭雪峰1, 崔钧贺2, 余启明1, 张磊1, 王娜1
作者信息
  • 1.桂林医学院公共卫生学院，广西 桂林 541000
  • 2.东北大学生命科学与健康学院

    • 尹紫月（1997—），女，硕士在读，研究方向：环境流行病学、精神流行病学

通讯作者:

王娜，E-mail：
Generalized anxiety disorder and plasma exosome-specific miRNA sinduced by drinking- water arsenic exposure
Zi-yue YIN1, Lin-nan GUO1, Wei-xing LEI1, Xue-feng GUO1, Jun-he CUI2, Qi-ming YU1, Lei ZHANG1, Na WANG1
Affiliations
  • School of Public Health, Guilin Medical University, Guilin, Guangxi 541000, China
出版时间: 2025-02-25 doi: 10.20043/j.cnki.MPM.202410452
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摘要
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目的

了解饮水型砷暴露致广泛性焦虑障碍 (Generalizedanxietydisorder, GAD) 的作用，探索血浆外泌体miRNAs的特异性改变。

方法

将16只SPF级wistar雄性大鼠随机分到正常组和染砷组，每组8 只，染砷组饮用水中的NaAsO2浓度0.10 mg/L，两组均可自由饮水。共染毒21天，分别用旷场和高架十字迷宫实验检测染毒的第0、7、14、21天的行为学，以评估GAD样行为；实验结束后，提取两组大鼠的血浆外泌体miRNAs进行测序，并运用实时荧光定量PCR验证差异miRNAs的表达情况。

结果

染毒后的第 21 d染砷组的体重低于正常组 (t=7.950，P<0.001) 。旷场实验结果显示，与正常组相比，染砷组的总路程减少 (t=2.213，P=0.044) ，修饰次数增加(t=-4.704，P<0.001)、跨格次数(t=4.340，P=0.001)和站立次数减少 (t=4.496，P=0.001)；高架十字迷宫实验结果显示，与正常组相比，染砷组的开臂次数 (t=3.614，P=0.003) 和开臂时间(t=4.775，P<0.001)减少，闭臂次数(t=-2.486，P=0.026)与闭臂时间增加 (t=-6.862，P<0.001)，砷暴露引起了大鼠GAD 样行为。实时荧光定量 PCR 结果显示，与正常组相比，染砷组中的miR-99b-3p 、miR-9a-5p 、miR-218a-5p 表达均上调；miR-425a-3p 、miR-378a-3p 、miR-155-5p 表达均下调（P<0.05）。GO 、KEGG基因富集分析结果显示，差异血浆外泌体miRNAs可能通过内吞作用、MAPK通路、Ras信号通路、cAMP信号通路、mTOR信号通路、凋亡来参与GAD的发生发展。

结论

饮水型砷暴露可以引起大鼠的GAD样行为，外泌体miR-99b-3p、miR-9a-5p、miR-218a-5p、miR-425a-3p、miR-378a-3p、miR-155-5p可能参与调节饮水型砷暴露致GAD的过程。

饮水型砷暴露  /  广泛性焦虑障碍  /  血浆外泌体miRNAs
Objective

To examine the impact of drinking water arsenic exposure on the development of generalized anxiety disorder (GAD) and to investigate specific changes in plasma exosomal miRNAs.

Methods

Sixteen male SPF-grade Wistar rats were randomly divided into two groups: control and arsenic. Each group consisted of 8 rats. The arsenic group was exposed to 0.10 mg/L. All groups had unrestricted access to drinking water. The rats were exposed to arsenic for a duration of 21 days. Behavioral assessments using the open field and elevated plus maze tests were conducted on days 0, 7, 14, and 21 to evaluate GAD-like behavior. At the end of the experiment, plasma exosomal miRNAs were extracted from both groups of rats. Sequencing was performed, and the expression levels of differentially expressed miRNAs were validated using real-time quantitative PCR.

Results

On the 21st day after arsenic exposure, the arsenic group exhibited lower body weight compared to the control group (t=7.950, P<0.001). In the open field test, the arsenic group showed a decrease in total distance traveled (t=2.213, P=0.044), an increase in rearing frequency (t=-4.704, P<0.001), an increase in grid crossings (t=4.340, P=0.001), and a decrease in standing frequency (t=4.496, P=0.001) compared to the control group. In the elevated plus maze test, the arsenic group exhibited a decrease in the number of entries into the open arms (t=3.614, P=0.003) and a decrease in time spent in the open arms (t=4.775, P<0.001), while showing an increase in the number of entries into the closed arms (t=-2.486, P=0.026) and an increase in time spent in the closed arms (t=-6.862, P<0.001) compared to the control group. These results indicate GAD-like behavior in the rats exposed to arsenic. Real-time quantitative PCR revealed that the expression levels of miR-99b-3p, miR-9a-5p, and miR-218a-5p were upregulated, while the expression levels of miR-425a-3p, miR-378a-3p, and miR-155-5p were downregulated in the arsenic group compared to the control group (P<0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed plasma exosomal miRNAs indicated their potential involvement in GAD development through processes such as endocytosis, MAPK pathway, Ras signaling pathway, cAMP signaling pathway, mTOR signaling pathway, and apoptosis.

Conclusion

Drinking water arsenic exposure can induce GAD-like behavior in rats. Plasma exosomal miRNAs, including miR-99b-3p, miR-9a-5p, miR-218a-5p, miR-425a-3p, miR-378a-3p, and miR-155-5p, may be involved in the regulation of GAD development induced by drinking water arsenic exposure.

Drinking water type arsenic exposure  /  Generalized anxiety disorder  /  Plasma exosome miRNAs
尹紫月, 郭林楠, 雷炜星, 郭雪峰, 崔钧贺, 余启明, 张磊, 王娜. 饮水型砷暴露引起广泛性焦虑障碍及血浆外泌体特异性 miRNAs研究. 现代预防医学, 2025 , 52 (4) : 622 -628 . DOI: 10.20043/j.cnki.MPM.202410452
Zi-yue YIN, Lin-nan GUO, Wei-xing LEI, Xue-feng GUO, Jun-he CUI, Qi-ming YU, Lei ZHANG, Na WANG. Generalized anxiety disorder and plasma exosome-specific miRNA sinduced by drinking- water arsenic exposure[J]. Modern Preventive Medicine, 2025 , 52 (4) : 622 -628 . DOI: 10.20043/j.cnki.MPM.202410452
正文
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广泛性焦虑障碍 (Generalized anxiety disease，GAD) 是焦虑障碍(anxiety disorders)的一种，以过分警觉、自主神经功能兴奋和持续明显的紧张不安等情绪表现为主，伴有眉头紧锁、面肌扭曲、姿势紧张等特征性行为改变[1]。且有研究发现，GAD的发生与脑卒中、血脂异常呈正相关[2]，反复发作的GAD患者可能出现记忆功能下降、人格功能改变、一定的自杀倾向，严重影响到了患者的生活质量。砷是一种神经毒物，近年来有研究发现，约20%的地方性砷中毒病人有神经症状，抑郁占8.47% 、焦虑伴抑郁占4.61%[3]。在前期对巴彦卓尔市开展的流行病学调查显示，水砷暴露、砷中毒均增加人群患GAD的风险[4]。动物实验结果也显示，亚慢性砷暴露可引起正常小鼠焦虑样行为[5]。并且通过饮用水摄入砷是人群摄入砷的主要途径之一[6]，因此，饮水型砷暴露引起的机体神经系统损伤受到了全球范围内的普遍关注。
外泌体 (Exosomes) 是细胞分泌的一种胞外微小包囊 ( small extracellular vesicle，sEV)，内含丰富蛋白质、DNA、mRNA、miRNA等，可在细胞间进行物质传递，并参与多种神经系统疾病病理学过程[7]。它广泛存在于血液、尿液、唾液、脑脊液和乳汁等体液中。并且外泌体可通过血脑屏障，此外，血浆和脑脊液中的外泌体含量可反映持续的神经过程[8] ，可以作为神经系统疾病发生发展的生物标志物。而与血浆、唾液或其他生物液体相比，外泌体miRNA高度丰富[9]。有研究表明外泌体miRNA异常对神经系统疾病，如抑郁[10]、帕金森[11]等有影响。
本研究拟通过动物实验验证饮水型砷暴露引起大鼠GAD样行为，检测血浆外泌体miRNAs的差异表达，进一步分析血浆外泌体miRNAs发挥作用的信号通路。为砷暴露引起GAD的机制探索提供思路，为GAD的预防提供依据。
1　对象与方法
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1.1　实验对象及伦理声明
健康雄性Wistar大鼠30只，SPF级，体重 (355.18±6.60) g,购买于广州维通利华实验动物技术有限公司，动物合格证号：NO.3359754033 。该研究得到了桂林医学院伦理委员会的批准 (GLMC202103366) 。
1.2　实验设计
30只大鼠在实验开始之前，预养1周，接着进行旷场实验，记录每只大鼠的水平得分和垂直得分，并相加总得分。取评分相近的大鼠16只，按照随机数字表随机分为正常组和染砷组，每组8只大鼠，同组同笼喂养。饮用水中NaAsO2含量依据根据中国饮用水标准GB 5749-2022，饮用水中无机砷的限值为0.01 mg/L（10μg/L），水中的NaAsO2浓度超过标准限制值的10倍称为高砷饮水[12]，可造成严重的砷中毒情况，因此将染砷组大鼠饮用的水中NaAsO2的含量设置为0.10 mg/L，正常组为自来水，两组均可自由饮水。给予每只鼠每天定量饲料25 g，每日喂食时记录每笼剩余粮量。实验期间进行体重的测量，染毒21 d后麻醉、腹主动脉采血，提取血浆外泌体进行miRNAs测序，并通过实时荧光定量PCR进行验证，通过GO和KEGG基因富集分析差异miRNAs调控GAD可能的信号通路。
1.3　实验方法
1.3.1　行为学测试
于染毒的7 d、14 d、21 d三个时点，进行旷场实验(openfield test，OFT) 和高架十字迷宫 ( elevatedplus-maze，EPM) 实验检测两组大鼠GAD样行为。
1.3.2　血浆外泌体分离
染毒实验结束后，选用含有EDTA的抗凝管对wistar大鼠进行腹主动脉采血。采血结束后，室温静置30 min后，先以1 000~2 000×g，20 ℃离心10 min，提取上清至新管；室温，10 000×g离心20 min；转移上清至新管，加入1/3体积的RiboTMexosomeIsolation reagent(forplasmaorserum) ；颠倒混合，直至完全混匀样本；放入4 ℃冰箱静置30 min；4 ℃ ，15 000×g离心2 min；小心地吸去上清，沉淀即为血浆外泌体，保存于-80 ℃。
1.3.3　miRNAs的测序及分析
根据上述步骤，提取对照和染砷组大鼠的血浆外泌体，送上海伯豪生物技术有限公司进行纯化和测序分析；使用QIAseq miRNA试剂盒提取血浆外泌体miRNAs后用Qubit®2.0荧光计定量，并于IlluminaNovaSeq 6000上进行测序。筛选出差异表达的miRNAs。使用edgeR对两组差异的miRNAs进行分析，得到P<0.05，Fold-change >2的miRNAs。
1.3.4　实时荧光定量PCR
使用TRIzol试剂从血浆外泌体中提取总 RNA。利用NCBI网站（https://www.ncbi.nlm.nih.gov/）查询miRNAs序列设计引物，并由上海生工生物工程股份有限公司合成引物。miRNAs的表达情况用 2-ΔΔct方法进行相对定量分析，所有miRNA均以U6为内参基因。
1.3.5　差异miRNAs靶基因的GO、KEGG富集分析
利用miRTarBase、miRanda数据库，预测两组差异的miRNAs的靶基因，利用DAVID（https://david.ncifcrf.gov/）数据库对靶基因进行基因组百科全书（KEGG）和基因本体（GO）富集分析，对差异miRNAs调控的靶基因进行功能富集。使用“微生信”（http://www.bioinformatics.com.cn/）在线工具对图片进行绘制。GO 富集分析包括三个部分，生物过程（BP）、细胞成分（CC）和分子功能（MF）。
1.4　统计学分析
用SPSS 28.0 软件进行统计分析。数据正态性检验采用Kolmogorov-Smirnov 检验。计量资料若服从正态分布，采用采用来描述；采用t检验对两组同一时间的体重、行为学得分进行比较、miRNAs进行差异分析。若资料不服从正态分布，用秩和检验进行比较。检验水准α=0.05。
2　结果
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2.1　大鼠的一般情况
大鼠染毒21 d后，对两组大鼠的体型、精神以及活动状态等进行了分析，与正常组比较，染砷组大鼠饮水、饮食量减少，精神萎靡、情绪低落、反应迟钝，皮毛光泽度降低。从图1可观察到每周饲料平均剩余情况，造模开始前预养周，每笼大鼠均未产生饲料剩余情况。造模期间，正常组均未产生饲料剩余，但染砷组开始出现饲料剩余，以第二周剩余最为明显，平均每只剩余2.3 g。
2.2　大鼠体重变化
对第0/7/14/21 d两组大鼠进行t检验，染毒前，两组大鼠的体重基本相同，染毒21 d ，两组大鼠体重差异有统计学意义，结果显示染砷组大鼠的平均体重高低于正常组（见表1）。从图2也能观察到，随着染毒时间推移，两组大鼠的体重虽然均增加，但与正常组相比，染砷组大鼠的体重增长缓慢。
2.3　各组旷场实验结果比较
染毒21 d后，对两组大鼠旷场实验结果进行t检验，结果显示与正常组相比，21 d染砷组大鼠的总路程、跨格次数、站立次数均减少和修饰次数增加，见表2。
2.4　各组高架十字迷宫实验结果比较
染毒21 d后，对两组大鼠高架十字迷宫实验结果进行t检验，与正常组相比，染砷组的开臂次数和开臂时间减少，闭臂次数与闭臂时间增加，见表3。
2.5　血浆外泌体的形态鉴定
血浆分离物在透射电镜下呈现出球形扁平膜囊、伴有“茶托”样，为外泌体的经典外形。纳米颗粒跟踪分析（NTA）结果显示，分离物粒径分布在30~150 nm之间，且在110 nm处有粒径分布峰值，显示分离物大小较为均一。进一步提示，外泌体提取试剂盒可成功分离出血浆外泌体，且粒径集中在110 nm左右。见图3A/B。
2.6　血浆外泌体miRNAs测序及结果分析
通过二代测序结果可知，在正常大鼠的血浆外泌体和染砷大鼠的血浆外泌体中miRNAs的种类分别是566种和377种，其中共同表达的miRNAs有216种，见图4A。差异表达的有471种。并将两组外泌体中差异较大的55个miRNAs制成热图。见图4B。对差异倍数最大的6 个miRNAs（miR-99b-3p 、miR-9a-5p 、miR-218a-5p、miR-425a-3p、miR-378a-3p、miR-155-5P）进行q-PCR验证。见图4C。
2.7　GO、KEGGpathway基因富集分析
通过采用miRTarBase、miRanda这两个数据库，对上述有明显差异的6个miRNAs进行靶基因预测，再对这6个交集中的靶基因再取并集，共有662个靶基因。见图5A。并对其进行GO、KEGG 分析，结果显示，生物学过程中GO富集主要为化学突触传递和对外来神经刺激的反应等；细胞组分GO富集主要为质膜整体成分、神经元反应等；分子功能GO富集主要为经递质受体活性、多巴胺神经递质受体活性等。KEGG富集分析气泡图可知，这 6个差异性血浆外泌体miRNAs可以通过内吞作用、MAPK通路、Ras信号通路、cAMP信号通路、mTOR信号通路、凋亡参与GAD 的调控。见图5B/C。
3　讨论
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砷是大自然中常见的一种对人体健康具有损害作用的重要类金属元素、环境毒物和已确认的人类致癌物[13]。本研究中，采用含亚砷酸钠(0.10 mg/L)的饮用水喂养健康雄性 Wistar 大鼠，采用旷场和高架十字迷宫实验检测到染砷组大鼠表现出GAD样行为。证实了饮水型砷暴露会引起焦虑样行为的改变。但其中的具体的发病机制尚不清楚，有待进一步探索。
外泌体广泛分布于许多体液中，且可以在细胞间传递生物信息[14]。本研究通过生物信息学分析发现，在正常和染砷组血浆外泌体中有 471 种差异表达的miRNAs ，对差异倍数最大的6个miRNAs 进行 q-PCR验证，结果显示miR-99b-3p 、miR-9a-5p 、miR-218a-5p 表达上调；miR-425a-3p、miR-378a-3p、miR-155-5P表达下调。对上述差异表达的 miRNAs进行 GO、KEGG分析可知，外泌体miRNAs 可能通过内吞作用、MAPK通路、Ras信号通路、cAMP信号通路、mTOR信号通路、凋亡等调控GAD的发生发展。
有研究发现，miR-99b-3p在胃癌[15]、肾细胞癌[16]等癌症中发挥着调节作用；miR-9a-5p可通过炎症反应、细胞增殖和凋亡等过程参与癫痫的发作[17]；miR-218a-5p的过表达与大鼠急性胆汁淤积性肝损伤有关[18]；临床研究发现，miR-425a-3p可通过MAPK/Wnt信号通路参与重度抑郁症的发生[19]；检测阿尔兹海默症患者血清外泌体miRNAs发现，miR-378a-3p表达失调并通过调节细胞衰老参与此疾病的发生发展[20]；miR-155-5P通过调节SKP2/IKKβ轴参与阿尔兹海默症的发病[21]。但尚未见以上miRNAs或外泌体miRNAs与GAD的关系，因此，深入探讨差异血浆外泌体miRNAs与GAD的关系具有重要价值。而对以上通路研究发现，检测焦虑和抑郁症失调的蛋白组，发现5-HT受体和 SERT内吞作用在焦虑和抑郁症的发生发展中起着重要的作用[22]，与本研究生信分析结果相一致；有研究表明，依兰精油[23]可以改变 ERK1/2和cAMP反应元件结合蛋白的磷酸化水平，并通过调节小鼠MAPK通路来逆转 m-CPP 产生的焦虑行为；RC3是一种突触后蛋白，在空间学习和焦虑情绪中起着重要的作用，它可以通过抑制Ras-ERK1/2信号轴，负向调节海马神经元分化[24]；高脂饮食诱导的肥胖可能通过抑制AMPK、促进mTOR磷酸化以抑制自噬而导致小鼠出现抑郁和焦虑样行为[25]；越来越多的证据表明焦虑症与睡眠剥夺之间存在关联，褪黑素可以通过改善细胞凋亡来减轻焦虑样行为[26]。以上通路均可能调控GAD的发生发展。
综上所述，研究结果表明饮水型砷暴露可导致GAD的发生，染砷组血浆外泌体与正常组相比，共有6个差异表达的 miRNAs。对相应的靶基因进行功能富集可知，差异血浆外泌体 miRNAs 可能通过内吞作用、MAPK 通路、Ras信号通路、cAMP信号通路、mTOR信号通路、凋亡通路调控GAD的发生发展。本研究通过动物实验验证饮水型砷暴露确实可以引起GAD的发生，进一步从动物层面上探讨血浆外泌体miRNAs调控GAD发生的潜在机制。但上述血浆外泌体miRNAs及相关的通路是如何参与GAD的调控机制还有待进一步探索与验证。
基金
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  • 国家自然科学基金(82160642)
  • 广西自然科学基金(2021GXNSFBA196014; 2024JJA140974)
文献
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2025年第52卷第4期
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doi: 10.20043/j.cnki.MPM.202410452
  • 接收时间：2024-10-29
  • 首发时间：2026-03-18
  • 出版时间：2025-02-25
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  • 收稿日期：2024-10-29
基金
国家自然科学基金(82160642)
广西自然科学基金(2021GXNSFBA196014; 2024JJA140974)
作者信息
    1.桂林医学院公共卫生学院，广西 桂林 541000
    2.东北大学生命科学与健康学院

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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
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红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
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